Experimental Determination of the Ion Selectivity of an ATP-Synthase Membrane Rotor by Isothermal Titration Calorimetry

ATP synthases are rotating nanomachines that couple ATP synthesis or hydrolysis to the transmembrane flow of protons or sodium ions down or against their electrochemical gradient. The key coupling element is a membrane-embedded subcomplex, the c-ring. We have recently proposed a principle that explains the ion selectivity of the c-ring, and validated this through functional studies of several ATP synthases. Specifically, we have proposed that a conserved Glu/Asp confers a universal H+ selectivity to all c-ring binding sites, and that additional amino-acids, which vary among species, have evolved to modulate this selectivity. In particular, we have shown that polar groups can suppress the H+ selectivity of the c-ring by a factor of 1-103. Thus, the enzyme becomes coupled to Na+, due to the large excess of Na+ over H+ under physiological conditions. Here, we further assess this theory by directly measuring the selectivity of a representative c-ring through Isothermal Titration Calorimetry. Specifically, we characterized the c-ring from the ATP synthase of Ilyobacter tartaricus. From titrations at different pH values, we established that Kd(Na+) ∼ 0.3 mM while Kd(H+) ∼ 0.3 μM, confirming the notion that this prototypical Na+-coupled c-ring is in fact H+ selective, although to a much smaller degree than those actually H+-driven. Comparing our results with those obtained for Enterococcus hirae demonstrates that, as predicted by our theory, the I. tartaricus c-ring is 100 times less Na+ selective. The weaker affinity for Na+ of the I.tartaricus ring is also coherent with the 10-fold difference in Km(Na+) values between these enzymes, at high pH. Taken together, these experiments demonstrate that the c-ring is the main determinant of the physiological ion specificity of rotary ATPases, and provide a conclusive validation of our theory.